CN110468150A - RGS1 gene is improving widow according to the application under environment in tomato bacterial leaf spot resistance as negative regulatory factor - Google Patents

Abstract

The invention discloses the application of the RGS1 gene as a negative regulatory factor in improving the resistance of tomato bacterial leaf spot in a low-light environment. The nucleotide sequence of the protein coding region of the RGS1 gene is shown in SEQ ID NO.1. The approach of the above application is to improve the bacterial leaf spot resistance of tomato mutants in a low-light environment by knocking out the RGS1 gene. The present invention uses CRISPR/Cas9 gene editing technology to obtain a mutant of tomato RGS1 gene editing, and finds that the mutant can not only significantly enhance the resistance to tomato bacterial leaf spot, but also significantly reduce tomato bacterial leaf spot in a low-light environment The occurrence degree of the RGS1 gene proved the use of RGS1 gene as a negative regulator in alleviating the occurrence of tomato bacterial leaf spot disease under the low-light environment, and can be used for the breeding of bacterial leaf spot resistant varieties under the low-light environment.

Description

RGS1 gene acts as a negative regulator to enhance tomato bacterial leaf spot under low light conditions applications in disease resistance

technical field

The invention relates to the field of biotechnology, in particular to the application of the RGS1 gene as a negative regulatory factor in improving the resistance of tomato bacterial leaf spot in a low-light environment.

Background technique

From 2000 to 2015, the sown area of vegetables in my country experienced a stage of rapid growth. In recent years, there is still a slow growth trend, but it remains basically stable. At present, vegetables have become the economic crops with the largest degree of marketization in my country. According to the 2018 China Statistical Yearbook data, the proportion of vegetable planting in the crop planting structure increased from 9.75% in 2000 to 12.0% in 2017, ranking first among all kinds of economic crops (http://www.stats.gov.cn /tjsj/ndsj/2018/index.htm). Among them, tomato is widely loved because of its rich nutrition and delicious taste. At the same time, tomato is also one of the main vegetables in protected cultivation. With the continuous change of the atmospheric environment and the facility-based agricultural planting methods, the lack of sunshine caused by unfavorable weather conditions such as smog and continuous rain and shaded by facilities is becoming more and more serious. Tomato is a light-loving plant. When the light intensity is lower than 150umol m ^-2 s ^-1 , tomato plants tend to grow excessively and the number of flowering decreases. Especially in the low-light environment, the outbreak of tomato diseases is serious, and various bacterial, viral and fungal diseases occur frequently and are very rampant. The common hazards of lack of light and diseases, coupled with a single planting model, have posed a great threat to the yield and quality of protected tomato cultivation.

Bacterial leaf spot is one of the high incidence diseases, caused by Pseudomonas syringa epv.tomato DC3000, which is a Gram-negative bacterium. The harm of this pathogen to tomato is mainly manifested on the leaves, and can also occur on the veins and fruits, affecting the fruit quality and yield of tomatoes. The outbreak of bacterial leaf spot of tomato was more serious in low-light environment, which caused greater economic losses to farmers.

On the one hand, the use of chemical pesticides to control bacterial leaf spot is still the most common means in China. Although chemical pesticide control has the advantages of simple use, high efficiency, and quick results, there are also serious problems: 1. Long-term use of a certain type of pesticide will cause harmful organisms to develop certain resistance, and the control effect will gradually decline; 2. 1. A large amount of chemical pesticide input has seriously damaged the agricultural ecological balance. The number of natural enemies of some pests has decreased due to the impact of pesticides, and harmful organisms may develop certain resistance, and it is very likely that harmful organisms will become rampant again; 3. The use of pesticides not only affects Environmental ecology, residual chemical pesticides will also cause food safety problems and pose a threat to people's health.

On the other hand, in a low-light environment, supplementary light measures can be used to increase light intensity and reduce diseases. However, supplementary light needs to build equipment, which will consume a lot of manpower, material resources and funds. Moreover, it is generally more aimed at facility vegetable cultivation, and is not suitable for solving the problem of lack of sunlight in large-scale open field cultivation. Fundamentally solve the increasingly serious problem of underprivileging.

Taking the above two considerations into consideration, finding new, effective, ecologically green, and economically harmless methods to effectively improve the ability of tomatoes to cope with frequent diseases in adverse environments such as low light is one of the problems that need to be solved urgently in vegetable production. , is also a research hotspot in today's vegetable production and scientific research. Breeding resistant cultivars that can enhance resistance to tomato diseases in low-light and other environments is a good solution and has important practical production significance.

In recent years, CRISPR/Cas9 gene editing technology has been developed rapidly. Compared with traditional breeding, this technology can accurately knock out any gene in the genome, thereby precisely changing the traits of crops, quickly obtaining ideal germplasm, and greatly shortening the breeding time. And the use of CRISPR/Cas9 gene editing technology for breeding can not introduce foreign genes, avoiding the controversial transgenic technology. Using this technology, it is possible to precisely knock out genes that negatively regulate diseases in some crops, and create and breed resistant varieties. Therefore, it is particularly important to find genes that negatively regulate diseases in crops.

Contents of the invention

The invention provides a new application of the RGS1 gene as a negative regulatory factor in improving the resistance of tomato bacterial leaf spot in a low-illumination environment, and for cultivating tomatoes with improved crop resistance, especially reducing the occurrence of bacterial leaf spot in a low-illumination environment. Variety provides the basis.

The specific technical scheme is as follows:

The present invention provides a new application of RGS1 gene as a negative regulatory factor in improving the resistance of tomato bacterial leaf spot in a low-light environment. The whole gene DNA sequence of the RGS1 gene is shown in SEQ ID NO.2, and the protein coding region is The nucleotide sequence is shown as SEQ ID NO.1, and the length of the protein coding region is 1434bp; the application method is to improve the bacterial leaf spot resistance of tomato mutants under low light environment by knocking out the RGS1 gene. The protein encoded by the RGS1 gene regulates the signal of the G protein and consists of 477 amino acids, and its sequence is shown in SEQ ID NO.3.

Under normal light conditions, plants form carbohydrates such as sucrose through photosynthesis, which are transported through the phloem to the intercellular space, that is, the apoplast region. Glucose and fructose are broken down in this area by sucrase. The RGS1 gene on the cell membrane can sense the apoplast glucose and undergo endocytosis, which weakens the interaction between the RGS1 protein and the G protein α subunit GPA1, causing the activation of the downstream G protein, thereby enhancing the plant's resistance to bacterial leaf spot. resistance.

However, it has been found through experiments that in a low-light environment, the external light intensity is weakened, and the photosynthesis of plants is weakened, resulting in a significant decrease in the glucose content of the tomato apoplast; the degree of endocytosis of the RGS1 protein encoded by the RGS1 gene is reduced by sensing the apoplast glucose signal , the RGS1 protein on the cell membrane still interacts with the G protein α subunit GPA1, resulting in the inability to activate the disease resistance process mediated by the downstream G protein signaling pathway. Based on the above experimental phenomena, the present invention uses gene editing technology to mutate the RGS1 gene, causing the G protein to be in an activated state, thereby enhancing the resistance of plants to bacterial leaf spot.

The present invention first carries out sequence analysis to tomato RGS1 (gene number: Solyc05g014160, PGSB website http://pgsb.helmholtz-muenchen.de/plant/tomato/), searches for the PAM sequence, and defines the sequence of 20 bp before NGG as sgRNA, select a highly specific sgRNA sequence positioned on the protein coding region of the gene, and the DNA sequence of the sgRNA specifically targeting the protein coding region of the RGS1 gene is shown in SEQ ID NO.4.

The present invention constructs the RGS1 gene CRISPR/Cas9 vector through gene editing technology and tissue culture technology, and screens to obtain a homozygous mutant strain rgs1#1 that is genetically stable and does not contain exogenous Cas9 protein. By inoculating the pathogenic bacteria of tomato bacterial leaf spot under normal light and low-light environment, and counting the incidence, it was found that rgs1#1 plants could significantly reduce the occurrence of tomato bacterial leaf spot disease under low-light environment.

Further, the low light environment is an environment where the light intensity is significantly weaker than the light intensity required for the proper growth of tomato, and the light intensity is <150umol m ^-2 s ^-1 , that is, the light intensity is >0umol m ^-2 s ^-1 and <150umol m ^-2 s ^-1 .

In addition, the present invention also finds that the glucose content of the apoplast of tomato leaves is significantly reduced after the tomato plants are placed in a low-light environment for 6 hours by liquid-phase HPLC; and by constructing an Agrobacterium vector of 35S:RGS1-GFP, the vector After the Agrobacterium liquid was injected into tobacco, the protoplasts were extracted to observe its subcellular localization, and it was found that the RGS1 gene was localized on the cell membrane (Fig. 4a).

The present invention also provides a method for alleviating tomato bacterial leaf spot in a low-light environment, comprising:

(1) cultivating tomato mutants resistant to tomato bacterial leaf spot in a low light environment;

(1-a) Select a target fragment containing a PAM structure in the protein coding region of the tomato RGS1 gene, and design corresponding primers based on the first 20 bases thereof to construct a CRISPR/Cas9 vector;

(1-b) constructing the Agrobacterium genetically engineered bacterium containing the CRISPR/Cas9 vector described in step (1-a);

(1-c) transforming tomato cotyledons with the genetically engineered bacteria described in step (1-b), to obtain a mutant that is genetically stable and does not contain exogenous Cas9 protein;

(2) Using the tomato mutant described in step (1) to replace the wild-type tomato planted in a low-light environment, the low-light environment is an environment where the light intensity is significantly weaker than the light intensity required for tomato growth, and the light intensity is <150umol m ^-2 s ^-1 . Further, in step (1-a), the nucleotide sequence of the first 20 bases of the PAM structure of the target fragment is shown in SEQ ID NO.4.

Further, the nucleotide sequences of the primers for constructing the CRISPR/Cas9 vector are shown in SEQ ID NO.5 and SEQ ID NO.6.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention utilizes the CRISPR/Cas9 gene editing technology to obtain the tomato RGS1 gene editing mutant, and finds that the mutant can significantly enhance the resistance to tomato bacterial leaf spot, and can significantly reduce the risk of tomato bacterial leaf spot disease in a low-light environment. The degree of occurrence proves the use of the RGS1 gene as a negative regulatory factor in improving the resistance of tomato bacterial leaf spot in a low-light environment, and can be used for the breeding of tomato varieties resistant to bacterial leaf spot.

Description of drawings

Fig. 1 is the gene editing site of the T1 generation mutant plant obtained in Example 2;

Compared with the non-gene-edited common tomato, the gene-edited mutant had a base deletion at the position of the sgRNA. Hereinafter, the common tomato without gene editing is called the control, and rgs1#1 has one base less than the control.

Fig. 2 is the histogram of the disease grade index of control and RGS1 gene mutant tomato inoculated with bacterial leaf spot pathogen under normal light environment and low light environment in embodiment 3;

Among them, the more severe the disease, the higher the disease grade index; the disease grade index of the control plants in the low-light environment was significantly higher than that of the normal light; the disease grade indexes of the control plants were significantly higher than those of the mutant materials; the mutation There is no significant difference between the disease grade index of the material under the low light environment and the disease grade index under the normal light, but slightly increased; the lowercase letters a, b, c represent significant differences at the 5% level between different plants.

Fig. 3 is the determination of the relative glucose content in the apoplast of tomato leaves after 6 hours of low-light environment treatment in Example 4; lowercase letters a and b represent significant differences at the 5% level between different treatments.

Figure 4 is the endocytosis of tomato RGS1 protein after exogenous treatment of different concentrations of glucose in Example 5;

Wherein, all the fluorescence observation results in Fig. 4 use the tobacco protoplast system. Figure 4a represents the subcellular localization of RGS1-GFP fusion protein without any treatment, located on the cell membrane. Figure 4b represents the endocytosis of RGS1-GFP after being treated with different concentrations of glucose for 30 min. The higher the concentration of glucose treatment, the more endocytosis.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the following enumerations are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the examples are well known to those skilled in the art, and the raw materials and kits used are commercially available.

The tomato variety used in the following examples is the conventional tomato variety CR (Condine Red), and a common tomato without gene editing was used as a control.

Example 1 Construction of CRISPR/Cas9 vector containing specific sgRNA

Find the DNA sequence of RGS1 (Solyc05g014160) on the PGSB website http://pgsb.helmholtz-muenchen.de/plant/tomato/, its sequence is shown in SEQ ID NO.1, enter http://crispr.hzau.edu .cn/cgi-bin/CRISPR2/CRISPR website, find out the 20bp base sequence TCAGAAAGAGACATTGGTGG (SEQ ID NO.4) located in front of a PAM structure in the protein coding region with high onscore score and GC content >40%.

Design CRISPR primers as follows:

CRISPR front primer (SEQ ID NO.5): GATTGTCAGAAAGAGACATTGGTGG;

Post CRISPR primer (SEQ ID NO.6): AAACCCACCAATGTCTCTTTTCTGAC;

Take 5 μl each of the front and back primers of the above CRISPR, mix well, and anneal with a PCR machine to form double strands. The intermediate vector pMD18-T was cleaved with BbsI, purified with a common DNA purification kit, then ligated with the vector with T4 ligase, and ligated overnight at 16°C. Heat shock at 42°C to transform the coated plate, resistance to ampicillin.

After some colonies of a certain size grow on the plate, single-clonal colonies can be picked, and PCR verification is performed using the CRISPR pre-primer (SEQ ID NO.5): GATTGTCAGAAAGAGACATTGGTGG and the vector post-primer (SEQ ID NO.7): CTACTTATCGTCATCGTCTTTG.

Send the bacterial solution with the correct band size to the sequencing company for sequencing. The sequencing results show that the vector contains sgRNA sequence, then the plasmid can be extracted from the bacterial solution, and after double digestion of the plasmid at the Hind III and Kpn I sites, it can be connected to On the final vector pCAMBIA1301. The re-sequencing results showed that the final vector contained sgRNA, and the resulting final plasmid could be electroporated into the competent GV3101 Agrobacterium. After culturing at a constant temperature of 28°C for two days, single-clonal colonies were selected for PCR verification to obtain a CRISPR/Cas9 gene editor that could be used to construct it. Material Agrobacterium strains.

Example 2 Preparation and Identification of RGS1 Gene Mutant Materials

The sterilized seeds were sown in the medium, and the cotyledons were cut after 7 days. Agrobacterium infection method The final plasmid prepared in Example 1 was transformed into cotyledons, and the totipotency of plant cells was utilized, and the cotyledons grew into complete plants to obtain the gene-edited tomato material of the T ₀ generation.

Detection of gene-edited tomato seedlings in T ₀ generation. Using the CTAB method to extract the genomic DNA of the T ₀ generation plants and use it as a template, design the following primers at about 200 bp before and after the DNA sequence containing the sgRNA, and perform PCR amplification and sequencing verification:

Primer before verification (SEQ ID No.8): TTTTAAGTGCATCGGTGAC

Primer before verification (SEQ ID No.9): GACTGGAAAGCAAGGAGG

The resulting PCR products were sent to a sequencing company for sequencing. Use DNAMAN software to compare the sequencing results with the original sequence of the gene. The sgRNA sequence has base deletions and insertions, and the plants that show a single peak are the required homozygous materials, which can be used for self-breeding to obtain T ₀ generation of seeds.

The T ₀ generation seeds were sown and placed in a growth chamber to obtain T ₁ generation plants. The same method as above was used to detect the base editing of the _sgRNA sequence in the T1 generation plants. At the same time, using the CRISPR pre-primer (SEQ ID NO.5) and vector post-primer (SEQ ID NO.7) to amplify the DNA _of the T1 generation plants by PCR, and detect whether the amplified product contains the Cas9 sequence. The selected detection results showed that the plants with sgRNA variation and no Cas9 were identified as T1 generation gene edited plants, named rgs1# ₁ , and their gene editing sites were shown in Figure 1.

rgs1#1 was one base less than the control plant, and the line was self _- propagated to obtain T1 generation seeds. After sowing, T2 generation plants with _sgRNA variation, no exogenous Cas9 gene and stable inheritance were obtained.

The following examples are all experimented with the above-mentioned homozygous line T ₂ generation plants as materials.

Example 3 Research on the disease resistance of RGS1 gene editing mutants under normal light and low light environment

Inoculate the bacteria of bacterial leaf spot pathogen onto the solid medium King's B containing 25 mg/L rifampicin, and cultivate them in an incubator at 28°C for 2 days to activate the bacteria, as the original plate. Pick the colony from the original plate, draw it on the new King's B medium, and culture it in the incubator at 28°C for 1 day. Suspend the bacterial liquid with 10 mM MgCl ₂ solution, adjust OD ₆₀₀ =0.1. Add 0.02% organic silicon and spray it on the back of the tomato plant leaves to allow the bacterial solution to infiltrate the leaves. Place the plants in an environment with a temperature of 25°C, relative air humidity of 95%, light and dark time of 12 hours each, light intensity of 450umol m ^-2 s ^-1 (normal light) and light intensity of 50umol m ^-2 s ^-1 (slightly illuminated) After 3 days of under-cultivation, the disease of the plants was observed.

The disease grade index was counted according to the incidence of the leaves, and the results shown in Figure 2 were obtained. The disease grade index of the control plant was significantly higher than that of the normal light environment under the low light environment; while the disease grade index of the mutant plants was not changed under the low light environment and under the normal light environment. Obvious; and the disease grade index of the mutant plants was slightly higher under low-light environment than under normal light.

The statistical method of the disease grade index is: grade the diseased tomato leaves, and the classification standard is: 0 means no disease, 1 means a few lesions can be seen on the lower epidermis of the leaves, 2 means local dense lesions on the lower epidermis of the leaves, and 3 Indicates that the lower epidermis of the leaf has dense lesions in many parts, and grade 4 indicates that the distribution of lesions can be seen on the entire leaf of the lower epidermis of the leaf. 0-4 grades are assigned 0-4 points respectively. According to the disease condition of each leaf of each plant, it is classified by grade, and weighted average is obtained to obtain the disease grade index of each plant.

It can be seen from Figure 2 that the RGS1 mutant can significantly reduce the occurrence of bacterial leaf spot in a low-light environment.

Determination of the glucose content of the apoplast after the embodiment 4 oligo-illumination environment treatment 6h

Put common tomato plants with consistent growth under the environment of light intensity 450umol m ^-2 s ^-1 (normal light) and light intensity 50umol m ^{- 2} s ^-1 (slightly illuminated), after 6 hours of treatment, extract 3g of tomato leaves Apoplast fluid. And by the method of HPLC liquid chromatography, measure the relative content of the glucose in its apoplast liquid.

It can be seen from Fig. 3 that the relative content of glucose in the apoplast decreased significantly after being treated for 6 hours under low-illumination environment, compared with that under normal light. It shows that the relative content of glucose in leaf apoplasts of tomato plants decreases under low light conditions.

Example 5 Endocytosis of Tomato RGS1 Protein After Exogenous Treatment of Different Concentrations of Glucose

First, using the control tomato cDNA as a template, the RGS1 protein coding region fragment (SEQ ID NO.2) was amplified and recombined into the pAC402-GFP vector, and the recombinant vector was electroporated into the competent GV3101 Agrobacterium to obtain a vector that could be used for transformation and infection. Agrobacterium strains. Then, the Agrobacterium strain was injected into tobacco, and the protoplasts were extracted 2 days later to observe the subcellular localization of the RGS1 gene.

It can be seen from Figure 4a that the RGS1 gene is localized on the cell membrane.

In addition, glucose solution was added to the above-mentioned fully extracted protoplast suspension to make the final concentrations of glucose 6%, 3%, and 1%, respectively. After 30 minutes of treatment, the subcellular localization was observed under a 40 times water microscope.

As shown in Figure 4b, after the glucose was processed, it was found that the green fluorescence of the RGS1-GFP fusion protein moved into the cytoplasm, that is, the RGS1 protein sensed the endocytosis of glucose. Moreover, as the concentration of exogenous glucose treatment increases, the more the green fluorescence moves into the cytoplasm, that is, the endocytosis is enhanced.

It can be seen from Figure 4 that glucose treatment can lead to the endocytosis of RGS1 protein, and this endocytosis has a concentration effect, the lower the glucose concentration, the less obvious the endocytosis. That is, the less glucose content in the apoplast, the less endocytosis of RGS1 protein on the cell membrane, and the weaker the activation of downstream G protein signaling.

sequence listing

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ttcacagaaa tgaaaagaag attgattacc tgcagatatt aaaggttgct caaaaatagt 1680

acaccacccc tgagataaag tgatgttata ttcctgttgg catgcccttc ctaaagatat 1740

gtcacaactg aattaaaatg aaacagaatt catctattat gtcttttcta tacttttttt 1800

ccttttcttc cttgtctctc tctctctctc tcatttactc ttttgtcgtt tgaaaaattt 1860

gagatatagc tttaaaagac tatttcttag tgctattgtt gattcatgtt acgtggaaaa 1920

actctttttc gatagtctta tatttctttg ttttgtatgg tgcttcagga gacgtctgcc 1980

accacttaga tcctatattt ttcttctgct gattctcttg ccatggatag ctcttgctgc 2040

aggtaagatt cttcaagttg aactctataa ataagtcttc ttttaagttt cttctattga 2100

aatactaaaa tatatattag tagatgaggg acttagggag ctggtggtgg aggttaggat 2160

gatgaaagac agggtattga cggtcaagct agtcaccgga gggcttactt taaacatgat 2220

tagtgcttac ggcggcaagt gggcttggag gagaagatca agaagtgctt ctgggaggat 2280

ttggatgagg tgttgaggga tatgccacat ttcaaaaagc tattcactgg cagagatttc 2340

aatggtcata ttgggtcaat cactagtggt tttgacagcggggatggagg ttttggtttt 2400

gggcttagga aaggaggagg agcatcactt cgggattttg ctaaagctta aggagttggcg 2460

ataaaaaatt catgcttccc aaaaaaggag gaccacatgg ccttggagat gggagagaag 2520

ttagagtcga tggaggcttg gggaagtagt ggagatgcaa ataacatgtg ggataagata 2580

gctagttgcg ttaggaaagt agtcagagag gtgttaggtt ttttcaggga ctatttttgc 2640

ggccatcaaa gggattggtg gtgaaatgga gatgtacaag gtatcatgaa aagcaaaaaa 2700

gttgctatgc taagttagtg ttgagaaaag atgaggtgga gaggcggaaa aacacgataa 2760

ggtataaggt gatgagaaag gacacgaagt agcatgggac cccagcctta gtgctgagat 2820

ttatacaaaa ccttgttacc tttgtcaaaa aaggaaagga cacgaagtag cagtcataac 2880

ggcaaaaacg acaactttca aacgcttgtt tgtcgatcta ggggacaaag aagaggataa 2940

gaagttgtac aggctcgcca aggcgagaga aaggaatcct cgggacttgg atcaagtgaa 3000

gtacatcaaa gatgaggacg gtaaagtact agtggaagag gcccacatta gacgaagagg 3060

gaaagcatac tttcataaac tcttgaacaa agaaagggtc acaaacattg tgttgggtgt 3120

tatagagaac tcggagagcg agatttgat tacggttggt gttttaaggt tgaagaggtt 3180

aagggtgcta tgtgtaagat gagtcagggt agagtcacta aactagatga attccggtag 3240

agttccaaaa aagcacaaaa agggcgggtt tagcgtggtt gcctaggttg tttaatgtca 3300

tttttaagac ctcaaaaaaa atattagtgt cccccttttt ttttaacgaa tgagcctctt 3360

ttacccattt aactacctac ttttatatgg gtaaaatgag tttgactcat tttttaccca 3420

ttttaaatga gttggatgcc aacatttaag ttgggtaatt tgaatgggta cccatataaa 3480

ttacttattc tgccacctct aaattatggg aggtgactca gagcacttac cagtcgagat 3540

ggggtcgcac caaggatctg cattgcacct gtttctattt gtcttggtga tggataagtt 3600

gatacggcat attctgcatg aggtgtcttg gtgtttgtta tttgtaaatg aattgtttt 3660

aattgaggag actcaggcgg agttaacgat tgattggagg tgttgagaca aatttgacaa 3720

tctaaatggt tcaagttgag ttggaccaag acagaagata tttgcctatt tggagtgtaa 3780

attcagtgat gtgacatagg caggggctga tccaaggttt aaaggctatg ggtgtcgtat 3840

cgccttttaa ataaacgtgt ttattaaatc aattaaatat ttaacctttt tcttaccttg 3900

taaataacct cagacttcag ttcgattatt cgacttttga gttttggaca aattaaatga 3960

attaaaaaaa agataatgac aacttggcgc aaaggaaaaa ttttccaata atatccttta 4020

taattacacc cgctgatcat attctaaata cacttaatga tggcataatt atttatgttt 4080

attaatactt cacgctctat gtaacatgct taataaaaag tggctaaatt tttttcatat 4140

acataatttt agtaacagaa acgaagtctt atgtacttta ttttgtttac attctcgata 4200

agatttaaat tatcataatc taaactattt ctttttatac attgaaatgt tttaataata 4260

gaacaaagag taagaaaaac tcggcgtcat gttttcagaa ttttgaccaa aagagaagat 4320

aattttggag gagaaggttt taagcaaaca ttaatcattt gtttgactat aaattatctc 4380

attaagggcc aaacaaacta aaattgtttc taatatacaa aagtagcatt cttttgtgga 4440

attgtggtag tctaaaaaag aaagtgtatc acgtgaattg agatgaagga aaaaaaagtt 4500

aacaaaataa aaattaaaaa gttttacaac atgttagcat atgtacatta tataatatag 4560

taaagtacat aaatatcatg tagtggccat ggcttaatgg ataaggtgat tcttaattgc 4620

agtgatatat gttggtttga aacatatcgg gaatcgtttt tgcaaaataa aattgttgca 4680

taaatggata actcaaagtg cacctatgcg gaattgaact cgcgtctttg acgacaacaa 4740

gttgacttgc caccagtgac aacatacgcc ccatttgtgc tgtgggtggc aagtttaata 4800

tttatataat ataatgtaca tatatacatg tcttacagaa tatcaatgga gatcattggg 4860

tgttgtggca cccccacggc tatgaataga tccgcccctg gtcgtaggag ccaagcatgg 4920

aaatgagact tgatactcaa gttatcccta agagcgagat ggagatattt aagtatctag 4980

ggtttttgct ccaaggaaat ggggagatca atgataatgt ctcaccgtat tggtgcggca 5040

tagatgaagt ggaggcttgc atctagtgtc ttgtgtgaca agaaggtacc accctaaatt 5100

aaaggtaagt tttatatagt ggtgtttaga ccgactttgg tgcatggggc gatgtgttgt 5160

ctagtcaaga actgccaggt tcagaagaag caagtggcag aaatgaggat attgagatgg 5220

atgtgcgggc atataggagc aatcatatta ggaatgagga tatctgacac gaagtggaag 5280

tggcttcttg gtggacaaga tgtggtggac aagatgaggg aagggagatt gagatatttt 5340

ggacacatga agaggagagg tgcggatgcc acaaaaagga ggtgtgagag gttggatata 5400

ggggttgagg agaggcagag gtaggccgaa gaagttttgt gaagaggtga ttagccaaga 5460

tatgccacat ttttagcgta ttgaggacat gaccttagat aggagggtac acgaataagg 5520

tagaagatta gtaggtgctg agagttgtct tattacttac ctattcatgt ctttggatgg 5580

gtgggagtat agagcgtcat gtgggttgta gtattagcat aacacatttag tctttagctt 5640

ttagtatctg attttatttg tggtttactg tgtttaggtg accgcacttt gctgctgctg 5700

ttacaattta cttgcatatc tgctaagcac tgctttgtta ttagctgtca tgtttttctt 5760

ttaattactg ttttgatttg ttggaaccga gagtgggtct gcgtacacta ccctaggttg 5820

ttactgttta cttagtagtt gctttcactg ggtatgttgt tgttgttgtg gaagtatgac 5880

ttgtcttttg cctacacttt tgcagttaag ctactttatg cattttaatt ttgtgtttta 5940

gtatttggta agtttatctt tcagttgagt ctgccaaaac tttgtgcaag tgtgtgaaca 6000

agtatttttt tactttcatt accatgtgac aatttgttga ttttagctca gtcaaattga 6060

ttttgataat cggcattaaa aattgctgaa aaattgcaaa aagtttgatt ttcaacactt 6120

atgtgattag tgtattctgc tcttttcata ttgtcacttc ggcacatgaa ttatctgtca 6180

tccctgatat taatattata agcttcattt gctaaagttg tttatgtaag aaaggatttt 6240

tgatattaat acggagtttg tctgatgaag ttattcagat aaaaaagcct ctgaacgacc 6300

ggtgccacat ggggacgctg tggatcatca ttgttatggg ccttcatgca ttatatgttg 6360

tagctttgat tgcttttgct ggggctgtgc atcatgtgga attcagattt catgaactca 6420

aagacctttg gagaggaatt cttgtctctt cagcttccat tggtttgttt ctatccattt 6480

tcttcgtttg gtttctttcc tggcatgctt ctgtgtcttc ctgtgtgtgc tttggtaggg 6540

gaatgacata ttaaggaatg aagtcagaaa gacatattta gtcttgatta acacttcctc 6600

aatattgttg tccaagcatc taaaggtatt cttacacata ccagaagcac gagcattata 6660

ctgaaatact gtcaaggaaa attcaatgga ggaaagtaac tagcttcagt gtaatggtca 6720

aagtggctga ctttcagatc taatagttga atttgacctt catgactgag acaccaattt 6780

aaaatacttc tcatgcattc ccctcaagtt ggataacatg catatcaaat cgcttacaga 6840

taaaattctt atcttaaatc tcagaaataa attgttatta attgcagctt gatatacagg 6900

gtttagataa tcacagataa caaaggcttc tagtatagga tctgtctata aagtataaac 6960

agtgagacat tttatacccc caatttaata caaaggaaag ttcaggaact acttggttaa 7020

acacaccaca ttatgtagat gtgagaactc aaatcaaagt ttttgatgga aattttaggt 7080

catgtaaatt gtgttgtagc cctggacaca agagtcttag cttggttcta ggaaagcatg 7140

ttatttattg ctttagcaag acttcgtttc ctcctagaga aaatttatac tcccttcact 7200

gtcacattta gcttcctgag aatcaaatag tgtgatattt gaccaagtct tttttgatat 7260

attttcattt ttgtaaggta taattttgaa tgtctaaatt ttaatctaag aaatgaaaat 7320

tctttatcca attatcaata gaattgatca aattgaccat aagcaaaact gtcaaccaaa 7380

ttgtatggga aactggggat gtatagagaa gtttttttcc tatgacttga gattcttgct 7440

gacagaaatg tatacactct tcattctatg tgaccagttg cttttgacac acccaaatgt 7500

gacgacaaaa cttttaaact gtgtattaga cacagttgag gaaaggtttg gtgtttcaaa 7560

gtttctttcg tgaagttagg gtgtgaaagg gatacagtga catgttttgg gggtgggtgg 7620

gtgggttaat tatttggcct atcttcaatc acaggaaaga aaatccttct atccatcaaa 7680

gcgtcattag caggattaac ttctagatct gacatttata agttggctat ataaagccct 7740

cctatgcttg agaaaaataa aaaattgaga ggcactttca acatgttttt gctgcctgtc 7800

tttttctata aaaaaaaaac acctccagtc agttgcttca cagtggcttt gttaactgaa 7860

gtatggttaa tggttaccat acttgataga aaggtgtaca agaagatgca atgaaagacc 7920

ctgatactcc gcatgatgtc aagttggctg caaaggacta ctcaagttct acccttatgc 7980

acactcatcc tgcctataaa aaaatcaagg ttctcctttg ccctcaacac cccaaaagtg 8040

cccttgatag tacaacttct ggagcatggt acttttgaag cacttgcgta aatctgaaga 8100

taatttattc caatcctcca acctcttaga tcgtggtact ggctacttat atcatatgtt 8160

gctcagagtc tccaaaatgt cgcctcactc ttgttgaatc cttcctaaaa tgcactactt 8220

ttggtattca tatacatttt tgaagagtct gagtaacata gcttatatcc ttgtattact 8280

ggggaacaga gatgtccggt ggtgttcgtc cttcctgccc ttccactatg tgatggtgca 8340

agttactaaa tttcatgggg tgaaaggact ctgattggcc tcatgtatcc tctttcatca 8400

tgatagatac aaccacctgc tctaagtaag aaaaggatag aaaattatgt tcagatatta 8460

acgctaggaa gttgtggttttatttttaaa tgtatgaaga catatgaaac tctgtaatat 8520

cgcaacttgt tttttatctt taatatttt tggtttgtta catcatgctt tattcaaata 8580

catatgttat gactttcatc tgtctgtttc aacattcact gttagttatact tgtagttcaa 8640

aactaaattg aaaatgatat ttcttcttag atatttcttc ttagacattt cccaagaata 8700

ttttgtgttg ataccagtct gttttctctg tatcaaggaa tatgggtggc tgcttatgta 8760

atgaatgagg ttcgcgaaga tatatcatcg ctagaaattg cctcaagatt cttattattg 8820

gttatggtac aatttcctga tcttgtgcta ctttgttgat atgtgagtat cagaatatgt 8880

ttgttgactg cctacagtttttctcttttt attcaacaga caagtgtcct tgtactggca 8940

ttcttctctt tctcgatttc tcaacctctt gtctcagtta tgagcttgag gaagaagaat 9000

cagaaagaat acaagacaat gagtcaggca ttaggtatac ctgatagtgg gatcctatta 9060

cagagggaat cgacaagcat tctagatcct aatgaacctt tggaaaagct cctgctgaat 9120

cgaaggttcc gtcagtcctt catggaattt gcagacaggt tttactcatt ctccttttgc 9180

aaaaatatca ttaaatcttc atgctgcgtg cgtgcatcag cttatcatgt gaagagagaa 9240

gatggtcctg tcttttctcc ttgattttaa cataattgaa cgtcaaaaga aataaaatgt 9300

atgtgatatt cttggaaaca aagaaataac agatataagc agaccaactt ttctagagag 9360

gaactctaac aagacaggtct gaaattgatt gcacagtaat agaatgacct ttttttcttt 9420

tactttgttc acatcgtagt tgtctggctg gagagagtgt gcacttctat gatgaagtgc 9480

aacattttga taaaattcct attcaggatt cagttaggag aatttacatg gcacgccaca 9540

taatagagaa gtatattgct gcaggtaagg tcaattcata tatgaagctt ttgatggtca 9600

tattaaatct tcccttcaaa gattctttaa agcaagtttt cttgggaact tgatggatta 9660

tacactttga tttttttttc tgtatcctcc ggtcctccct ctgaaaagta ttctcgtgct 9720

taaattttga tggttctctg acttgcattt gctcattaaa cacctgtagg agcaccaatg 9780

gaggtgaaca tttctcaccg aatccggcag gaaattttga atactaatga tctctcccac 9840

actgacctat tcaaaaacgc tctaggtgaa ctgatgcagc tgatgaaact ggttggtctt 9900

ttacattctg aaccattact tgcttgatta taatggtatt ttgcatattg cttagttatc 9960

ctctttctat ccttcccttcc gtctcactct catcatctct tgtactattg ttgaatcgtt 10020

cttcttgtgg acacacaaag aacttagcaa gagattactg gtcgtcaatg tacttcatga 10080

agctgcaaga ggatatccgc atgagagcag ttgatccgga gatggaacat gctagtggtt 10140

ggaatttttc cccaagattg agttctgtcc attgcagtga tgaccctttc cagaatgaac 10200

atacagaact gcgatgatga aacatgtgag tagggtcatg acttcccaac gtgatggagt 10260

tgcatatatt tggccgtaac aggaagagaa gggaacctaa acgagacatg atattcatct 10320

tcacaggatg ggctccagct acctgattct cctgttcata aacgatacga gccttcaaga 10380

ccaaattttg ctgtgtgatg ttgcacctca tgctttgcag acctga 10426

<210> 4

<211> 20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

tcagaaagag aattggtgg 20

<210> 5

<211> 25

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

gattgtcaga aagagacatt ggtgg 25

<210> 6

<211> 25

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

aaacccacca atgtctcttt ctgac 25

<210> 7

<211> 22

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ctacttatcg tcatcgtctt tg 22

<210> 8

<211> 19

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ttttaagtgc atcggtgac 19

<210> 9

<211> 18

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

gactggaaag caaggagg 18

Claims (5)

1. The application of the RGS1 gene as a negative regulatory factor in improving tomato bacterial leaf spot resistance in a low light environment, characterized in that the nucleotide sequence of the protein coding region of the RGS1 gene is as shown in SEQ ID NO.1 , the approach of the application is to improve the bacterial leaf spot resistance of tomato mutants under low-light environment by knocking out the RGS1 gene. 2 . The application according to claim 1 , wherein the low-light environment is an environment with light intensity significantly weaker than that required for proper growth of tomatoes, and the light intensity is <150umol m ^-2 s ^-1 . 3. A method for alleviating bacterial leaf spot of tomato under a low-light environment, characterized in that, comprising: (1) cultivating tomato mutants resistant to tomato bacterial leaf spot in a low-light environment; (1-a) Select a target fragment containing a PAM structure in the protein coding region of the tomato RGS1 gene, and design corresponding primers based on the first 20 bases thereof to construct a CRISPR/Cas9 vector; (1-b) constructing the Agrobacterium genetically engineered bacterium containing the CRISPR/Cas9 vector described in step (1-a); (1-c) transforming tomato cotyledons with the genetically engineered bacteria described in step (1-b), to obtain a mutant that is genetically stable and does not contain exogenous Cas9 protein; (2) Use the tomato mutant described in step (1) to replace the wild-type tomato planted in a low-light environment, the low-light environment is an environment where the light intensity is significantly weaker than the light intensity required for tomato growth, and the light intensity is <150umolm ^-2 s ^-1 . 4. the method for alleviating bacterial leaf spot of tomato under the low light environment as claimed in claim 3, is characterized in that, in step (1-a), the nucleotide sequence of the first 20 bases of the target fragment PAM structure As shown in SEQ ID NO.4. 5. the method for alleviating the bacterial leaf spot of tomato under the low light environment as claimed in claim 3, is characterized in that, construct the nucleotide sequence of the primer of described CRISPR/Cas9 carrier such as SEQ ID NO.5 and SEQ ID NO .6 shown.